Fluorosurfactants for stabilizing single-and double-emulsion droplets

Abstract

The invention provides a fluorosurfactant composition for use in stabilising emulsions, in particular emulsions comprising single or double-emulsion droplets; as well as improved methods for manufacture of the fluorosurfactant; wherein the surfactant comprises a perfluorocarbon chain amide covalently linked to a polymer of ethylene 5 oxide and propylene oxide. The invention also provides methods for single-cell resolution cell killing- and cell secretion-assays based on droplets stabilised by the fluorosurfactant composition.

1 . Method of synthesis of a fluorosurfactant having the formula: A-X-B or A-X-B-X-A, wherein:
 each instance of A is independently F—[CF(CF 3 )CF 2 O] c —CF(CF 3 )CONH—, wherein c is greater than or equal to 30;   X, is a covalent bond; and   each instance of B is independently —[C 3 H 6 O] d —[C 2 H 4 O] e —[C 3 H 6 O] f CH 2 CH(CH 3 )— or —[C 3 H 6 O] g —[C 2 H 4 O] h —CH 3 , wherein a combination of d and f is an integer greater than or equal to 2, e is an integer greater than or equal to 7, each of g and h are integers greater than 0 and a combination of g and h is an integer greater than or equal to 7;   comprising the synthesis step of reacting the carboxylic end group of a fluoropolymer having the structure F—[CF(CF 3 )CF 2 O] c CF(CF 3 )COOH with oxalyl chloride In the presence of N,N-Dimethylformamide to obtain an activated fluoropolymer intermediate having the structure F—[CF(CF 3 )CF 2 O] c —CF(CF 3 )COCl, wherein the activated fluoropolymer intermediate is subsequently reacted with a mono- or di-functionalized amine polymer of ethylene oxide (EO) and propylene oxide (PO) in a separate step.   
     
     
         2 . The method of synthesis according to  claim 1 , wherein the mono-functionalized amine polymer of ethylene oxide (EO) and propylene oxide (PO) has the structure:
   NH 2 —[C 3 H 6 O] g —[C 2 H 4 O] h —CH 3 .
   
     
     
         3 . The method of synthesis according to  claim 1 , wherein the di-functionalized amine polymer of ethylene oxide (EO) and propylene oxide (PO) has the structure:
   NH—[C 3 H 6 O]d-[C 2 H 4 O] e [C 3 H 6 O] f CH 2 CH(CH 3 )—NH 2 .
   
     
     
         4 . The method of synthesis according to  claim 1 , wherein the mono-functionalized amine polymer of ethylene oxide (EO) and propylene oxide (PO) has the structure:
   NH 2 —[CH(CH 3 )CH 2 O] g —[CH 2 CH 2 O] h —CH 3 .
   
     
     
         5 . The method of synthesis according to  claim 1 , wherein the di-functionalized amine polymer of ethylene oxide (EO) and propylene oxide (PO) has the structure:
   NH 2 —[CH(CH 3 )CH 2 O] d —[CH 2 CH 2 O] e —[CH(CH 3 )CH 2 O] f CH 2 CH(CH 3 )—NH 2 .
   
     
     
         6 . The method of synthesis according to  claim 1 , wherein c is an integer between 39 and 44, the combination of d and f is an integer between 3 and 7, e is an integer between 7 and 20, and the combination of g and h is an integer between 7 and 25. 
     
     
         7 . The method of synthesis according to  claim 1 , comprising the steps of:
 a. dissolving a species of the fluoropolymer having the structure F—[CF(CF 3 )CF 2 O]C—CF(CF 3 )COOH, wherein c is greater than or equal to 30, in a hydrofluoroether solvent,   b. activating the carboxylic end group of the dissolved species of fluoropolymer by reaction with oxalyl chloride in the presence of N,N-Dimethylformamide to form an activated Intermediate of said species,   c. removing unreacted oxalyl chloride by heating the reaction under reduced pressure,   d. reacting the activated intermediate of the species of fluoropolymer dissolved in dry hydrofluoroether solvent, with said mono- or said di-functionalized amine polymer of ethylene oxide and propylene oxide dissolved in an anhydrous mixture of dichloromethane and triethyamine, wherein the reaction is performed under N 2  atmosphere,   e. filtering the product of step (d) and concentrating the filtrate to provide an orange oil residue comprising the reaction products of step (d),   f. dissolving the orange oil residue In a 2:1 mixture of hydrofluoroether solvent and MeOH, and performing one or more rounds of phase separation to provide a phase enriched for the fluorosurfactant produced in step (d),   g. optionally filtering the phase enriched for the fluorosurfactant in step (f) and concentrating the filtrate,   
       wherein the hydrofluoroether solvent is preferably HFE-7100 or HFE-7500. 
     
     
         8 . A composition producible by the method according  claim 1 , comprising a fluorosurfactant having the formula: A-X-B or A-X-B-X-A, wherein:
 each instance of A is independently F—[CF(CF 3 )CF 2 O] c —CF(CF 3 )CONH—, wherein c is greater than or equal to 30;   X, is a covalent bond; and   each instance of B is independently —[C 3 H 6 O] d —[C 2 H 4 ] e —[C 3 H 6 O] f CH 2 CH(CH 3 )— or —[C 3 H 6 O] g —[C 2 H 4 O] h —CH 3 , wherein a combination of d and f is an integer greater than or equal to 2, e is an integer greater than or equal to 7, and wherein each of g and h are integers greater than 0 and a combination of g and h is greater than or equal to 7, wherein the composition comprising the fluorosurfactant having the formula: A-X-B or A-X-B-X-A has single-emulsion stabilization properties characterized by the formation of an emulsion comprising said composition, wherein less than 5% of the single-emulsion drops formed are coalescent droplets after at least 16 hours incubation at 30° C. and 10 min at 65° C. at about 1 atmosphere, wherein the coalescent droplets have an average cross-sectional dimension at least 2 times the average cross-sectional dimension of the observed droplets.   
     
     
         9 . A composition producible by the method according to  claim 1 , comprising:
 a fluorosurfactant having the formula: A-X-B-X-A, wherein:   each instance of A is independently F—[CF(CF 3 )CF 2 O] c —CF(CF 3 )CONH—, wherein c is greater than or equal to 30;   X, is a covalent bond; and   B is —[C 3 H 6 O] d —[C 2 H 4 O] e —[C 3 H 6 O] f CH 2 CH(CH 2 )—,   wherein a combination of d and f is an integer greater than or equal to 2, e is an integer greater than or equal to 7, wherein the composition comprising the fluorosurfactant having the formula: A-X-B-X-A has double-emulsion stabilization properties characterized by the formation of an emulsion comprising said composition, wherein less than 5% of the single-emulsion drops formed are coalescent droplets after six weeks of storage at room temperature and approximately 1 atmosphere of pressure, when droplet coalescence Is defined as a droplet having a diameter of at least 2 times the diameter of the average diameter of the observed double-emulsion droplets.   
     
     
         10 . The composition according to  claim 8 , wherein:
 A-X-B Is F—[CF(CF 3 )CF 2 O] c —CF(CF 3 )CONH—[CH(CH 3 )CH 2 O] g [CH 2 CH 2 O] h —CH 3      and,   A-X-B-X-A Is F—[CF(CF 3 )CF 2 O] c —CF(CF 3 )CONH—[CH(CH 2 )CH 2 O] d [CH 2 CH 2 O] e —[CH(CH 3 )CH 2 O] f CH 2 CH(CH 3 )NHCOCF(CF 3 )—[OCF 2 CF(CF 3 )] c —F.   
     
     
         11 . The composition according to  claim 8 , wherein c is an integer between 39 and 44, the combination of d and f is an Integer between 3 and 7, e is an integer between 7 and 20, and the combination of g and h is an integer between 7 and 25. 
     
     
         12 . The composition according to  claim 8 , wherein c is an integer between 40 and 43, the combination of d and f is an integer between 5 and 6, e is an integer between 12 and 13, g is an integer between 2 and 3 and h is an integer between 18 and 20. 
     
     
         13 . A mixed composition comprising a combination of the composition according to  claim 8 , wherein the fluorosurfactant has the formula: A-X-B; and the composition according to  claim 8  wherein the fluorosurfactant has the formula: A-X-B-X-A. 
     
     
         14 . The mixed composition according to  claim 13 , wherein the fluorosurfactant having the formula: A-X-B and the fluorosurfactant having the formula: A-X-B-X-A are combined in a ratio of between 1:5 and 1:20. 
     
     
         15 . An emulsion comprising:
 i. aqueous droplets dispersed in an oil continuous phase (single emulsion droplets) or   ii. oil-encapsulated aqueous droplets dispersed in an aqueous continuous phase (double emulsion droplets),   
       wherein said emulsion comprises the composition or mixed composition according to  claim 8 , and wherein the oil is a fluorocarbon. 
     
     
         16 . The emulsion according to  claim 15 , wherein the aqueous droplets comprise reagents for PCR or MDA amplification of a DNA molecule. 
     
     
         17 . The emulsion according to  claim 15 , wherein the aqueous droplets comprise one or more cells. 
     
     
         18 . A kit for generation of an emulsion, comprising a composition or mixed composition according to  claim 8 , in a first container, at least one microfluidic cartridge for forming single- and/or double-emulsion droplets, and a fluorocarbon oil in a second container. 
     
     
         19 . A method for forming stable double-emulsion droplets that are selectively permeable comprising: the formation of double-emulsion droplets of a water-in-fluorocarbon oil-in-water emulsion, wherein said fluorocarbon oil emulsion comprises the composition or mixed composition according to  claim 8 . 
     
     
         20 . A method for assaying the functionality of one or more cells comprising:
 encapsulating the one or more cells in double-emulsion droplets of a water-in-fluorocarbon oil-in-water emulsion, wherein said fluorocarbon oil emulsion comprises the composition or mixed composition according to  claim 8 .   
     
     
         21 . The method according to  claim 20 , wherein the method assays the functionality of one cell-type (the effector cell) acting upon another type (the target cell) comprising the steps:
 1) differential staining of the two cell-types,   2) encapsulate the two cell types in plural double-emulsion droplets forming an double-emulsion, incubate the emulsion of double-emulsion droplets, and   3) perform an analysis of the droplets by flowcytometry or imaging.   
     
     
         22 . The method according to  claim 20 , wherein the method is for assaying the functionality of one cell-type (the effector cell) acting upon another type (the target cell), comprising the steps:
 1) preactivation of effector cells,   2) differential staining of the two cell-types,   3) washing and resuspending cells in a suitable medium,   4) activate effector cells,   5) encapsulate the two cell types in plural double-emulsion droplets forming an double-emulsion, incubate the emulsion of double-emulsion droplets, and   6) perform an analysis of the droplets by flow cytometry or imaging.   
     
     
         23 . The method according to  claim 21 , wherein the effector cell is a CAR-T modified cell and the target cell is a cancer cell. 
     
     
         24 . The method according to  claim 20 , wherein the method is for assaying the functionality of a population of cells for excretion of a specific substance, comprising the steps:
 1) suspending the cells with an assay-composition which comprises:
 a. A reagent that binds to the surface of the cells and to the specific substance, and 
 b. An antibody that binds to the same specific substance and which is coupled to a fluorophore. 
   2) producing double-emulsion droplets containing cells and assay-components   3) incubating double-emulsion droplets in a CO 2  incubator   4) breaking the droplets to release the cells, and   5) analysing the released cells by flowcytometry or imaging.   
     
     
         25 . The method according to  claim 24 , wherein the specific substance is a specific interleukin. 
     
     
         26 . A method for obtaining a population of cells excreting a specific substance comprising:
 1) assaying a population of cells for excreting a specific substance using a FACS-instrument according to the method of claim  24  while setting the FACS-instrument to collect the population of cells excreting the specific substance, and   2) collecting the population of cells excreting the specific substance.